As described by at least Mitchell in Nat. Biotechnol. 19, 717-721 (2001), incorporated by reference herein for all purposes, microfluidic technology offers many possible benefits in chemistry, biology and medicine. One important possible benefit is to automate rote work while reducing the consumption of expensive reagents to the nanoliter or sub-nanoliter scale.
As described in Anal. Chem. 69, 3407-3412 (1997), incorporated by reference herein for all purposes, Hadd et al. developed a microchip for performing automated enzyme assay, in which precise concentrations of substrate, enzyme, and inhibitor were mixed in nanoliter volumes using electrokinetic flow. In Anal. Chem. 74, 2451-2457 (2002), incorporated by reference herein for all purposes, Fu et al reported an integrated cell sorter with a 1 picoliter minimum active volume by the actuated valve.
However, as pointed out by Meldrum et al. in Science 297, 1197-1198 (2002), incorporated by reference herein for all purposes, since microfluidic devices must at some point be interfaced to the macroscopic world, there is a minimum practical volume (of order—1 μl) that can be introduced into a device. The so-called “world-to-chip” interface problem described by Ramsey in Nat. Biotechnol. 17, 1061-1062 (1999), incorporated by reference herein for all purposes, has plagued the microfluidic field since its inception. As noted by Ross et al. in Anal. Chem. 74, 2556-2564 (2002), it is always questionable how the desired economies of scale in microfluidics can practically be achieved, unless an effective approach is developed to solve the mismatch between those two scales.
Although integrated glass capillaries have been used to reduce sample consumption for simple titration between two reagents, these devices are fundamentally serial and have the possibility of sample cross-contamination during the loading process. See Farinas et al., Analytical Biochemistry 295, 138-142 (2001), incorporated by reference herein for all purposes.
On the other hand, there has been an effort to develop techniques to concentrate analytes from a large input volume. In Anal. Chem. 73, 1627-1633 (2001), incorporated by reference herein for all purposes, Macounova et al. describe microfluidic isoelectric focusing (IEF) techniques. Ross et al. describe temperature gradient focusing (TGF) techniques. However, only a limited species of samples have successfully been demonstrated so far.
The challenge associated with realizing the desired economies of scale in microfluidic devices is to simultaneously reduce the number of pipetting steps needed to load the devices, while amortizing the sample volume from each pipetting step over a large number of independent assays. As pointed out in U.S. Pat. No. 6,508,988, incorporated by reference herein for all purposes, microfluidic matrix geometries offer the advantage of performing N2 independent reactions with only 2N pipetting steps.
In Lab on a Chip 2, 188-192 (2002), Kikutani et al. used N=2 matrices for chemical synthesis. In Anal. Chem., 73, 5207-5213 (2001), Ismagilov et al. used N=5 passive matrices to demonstrate two-component biochemical assays such as optical detection of enzymatic activity. However, in those devices the reagent consumption scaled only with N, which was so small that there were little practical savings.
Passive devices also have technical limitations in sample metering and device operation. For example, precise pressure balancing was required during operation of the devices of Kikutani et al., and the kinetics of mixing were limited due to the static nature in the devices of Ismagilov et al.
Accordingly, there is a need in the art for microfluidic techniques and apparatuses for addressing the “world-to-chip” interface problem.